(1) Field of the Invention
This invention relates generally to the regulation of apoptosis and to compositions therefor and, more particularly, to serine substituted mutants of BCL-X.sub.L /BCL-2 associated cell death regulator.
(2) Description of the Related Art
Programmed cell death, referred to as apoptosis, plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms (Raff, Nature 356:397-400, 1992 which is incorporated by reference). Genetic and molecular analysis from nematodes to humans has indicated that the apoptotic pathway of cellular suicide is highly conserved (Hengartner and Horvitz, Cell 76:1107-1114, 1994 which is incorporated by reference). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
Considerable progress has been made in identifying the molecules that regulate the apoptotic pathway at each level. Of note, both positive and negative regulators, often encoded within the same family of proteins, characterize the extracellular, cell surface and intracellular steps (Oltvai and Korsmeyer 1994 which is incorporated by reference).
One such family of proteins that constitutes an intracellular checkpoint of apoptosis is the BCL-2 family of proteins. The founding member of this family is the apoptosis-inhibiting protein encoded by the BCL-2 protooncogene which was initially isolated from a follicular lymphoma (Bakhshi et al. Cell 41:889-906, 1985; Tsujimoto et al, Science 229:1390-1393, 1985; Cleary and Sklar, Proc Natl Acad Sci USA 82:7439-7443, 1985 which are incorporated by reference). The BCL-2 protein is a 25 kD, integral membrane protein of the mitochondria. This factor extends survival in many different cell types by inhibiting apoptosis elicited by a variety of death-inducing stimuli (Korsmeyer, Blood 80:879-886, 1992 which is incorporated by reference).
The family of BCL-2-related proteins has been defined by sequence homology that is largely based upon conserved motifs termed BCL-homology regions. (Yin et al, Nature 369:321-323, 1994 which is incorporated by reference). BCL-homology regions 1 and 2 (BH1 and BH2 ) domains have been shown to be important in dimerization and in modulating apoptosis (Yin et al. Nature 369:321-20 323, 1994 which is incorporated by reference). A third homology region, BH3, has also been identified as important to dimerization as well as apoptosis (Boyd et al., Oncogene 11:1921-1928; Chittenden et al., Embo J 14:5589-5596, 1995 which are incorporated by reference) as has been a fourth homology region, BH4, near the amino terminal end of some family members (Farrow and Brown, Curr Opin Genet Dev 6:45-49, 1996 which is incorporated by reference).
Members of this family can heterodimerize and, in most cases, homodimerize as well. The ratio of death antagonists (BCL-2, BCL-X.sub.L, MCL-1 and A1 ) to agonists (BAX, BAK, BCL-X.sub.S and BAD) determines which homodimers or heterodimers are formed and the balance of these is believed to determine whether a cell will respond to an 35 apoptotic signal (Oltvai and Korsmeyer, Cell 79:189-192, 1994 which is incorporated by reference). Thus, dimerization between agonists and antagonists is competitive. For example, the death promoting molecule BAX forms homodimers that favor death whereas BAX will also form heterodimers with BCL-2 or BCL-X.sub.L (Oltvai et al. 1993 which is incorporated by reference) and the formation of these heterodimers results in inhibition of cell death.
BAD (BCL-X.sub.L /BCL-2 Associated Cell Death Regulator) is a death agonist (U.S. Pat. No. 5,622,852 which is incorporated in its entirety by reference), which is a very distant BCL2 family member in sequence homology, having only the BH1 and BH2 domains and lacking the BH3, BH4 and transmembrane anchoring domains. BAD apparently exerts its death promoting effects by heterodimerizing with BCL-2 or BCL-X.sub.L, death antagonists (Yang et al., 1995). Thus, BAD may function as an inducer of apoptosis by competing for the apoptosis repressors, BCL-2 and BCL-X.sub.L.
BAD does not homodimerize, lacks the typical signal/anchor segment suggesting that its free form may reside in the cytosol, has PEST sequences and a much shorter half-life. These parameters suggested BAD could prove to be more proximal at the BCL2 step perhaps linked to signal events. The BAD polypeptide might, therefore, function in a signal transduction role and provide a potential site for modulation of apoptosis.
The 14-3-3 family of proteins of which there are at least seven mammalian isoforms, are highly conserved and ubiquitously expressed. These proteins bind to and regulate a variety of proteins including a number of proteins involved in signal transduction. 14-3-3 binding has been shown to be sequence-specific to a phosphoserine containing motif (Muslin et al. Cell 84:889-896, 1996 which is incorporated by reference). This group identified two serine residues (Serine-259 and Serine-621) in Raf-1 each of which lies within a six amino acid motif and phosphorylation of these serines results in 14-3-3 protein binding at each of these two sites. Furthermore, this group identified a number of proteins that contained this motif and postulated that if appropriately phosphorylated, these other proteins might bind 14-3-3. One such protein was BAD and the motif identified surrounds Serine-136 of BAD. Nevertheless, this reference did not report on any tests to determine whether such binding indeed occurs. Furthermore, the physiologic role of any possible binding of a Serine-136 phosphorylated BAD to 14-3-3 was not considered by this group with only the general suggestion that 14-3-3 might interact with proteins to perform an essential chaperone function. Moreover, this group did not suggest any possible phosphorylation of any other of the Serine residues of BAD such as Serine-112.
Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals. Furthermore, some DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain other disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation. In such disease conditions it would be desirable to promote apoptotic mechanisms and one advantageous approach might involve treatment with a cell to modulate the effect of BAD to increase its binding to BCL-2 and/or BCL-X.sub.L and thereby diminish the expression of the death repressor activity of these death antagonists.
Conversely, in certain disease conditions it would be desirable to inhibit apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like. In the treatment of such diseases it would be desirable to modulate the effect of BAD to decrease its binding to BCL-2 and/or BCL-X.sub.L and thereby increase the death repressor activity of these death antagonists. Thus, it would be desirable identify new approaches that can alter the binding of BAD to BCL-2 family members and to utilize these as a basis for treatment modalities in advantageously modulating the apoptotic process in disease conditions involving either inappropriate repression or inappropriate acceleration of cell death.